From 1981 to date, there have been over thirteen thousand (13,000) people diagnosed as having acquired immune deficiency syndrome (AIDS). AIDS has been characterized by the onset of severe opportunistic infections secondary to an effect on the body's immune system. Gottlieb, MS. et al., Pneumocystis Carinic Pneumonia and Mucosal Condidisis in previously healthy homosexual men: evidence of a new acquired cellular immuno-deficiency, N. Eng. J. Med. 305, 1426-1431 (1981). The disease has been found in male homosexuals, patients receiving blood products, intraveneous drug addicts, and individuals originating from Haiti and Central Africa. Piot, P. et al, Acquired immunodeficiency syndrome in a heterosexual population in Zaire. Lancet 11, 65-69 (1984). The causative agent was suspected to be of viral origin as the epidemiological pattern of AIDS was consistent with a transmissible disease. At least three (3) retroviruses have been isolated from cultured T-cells of several patients with AIDS, or from white blood cells of persons at risk for the disease. A novel human retrovirus called lymphadenopathy-associated virus (LAV) was discovered and its properties were consistent with its etiological role in AIDS. That virus was isolated from a patient with lymphadenopathy and hence the name. Montagnier, L. et al. A New Human t-lymphotropic retrovirus: Characterization and possible role in lymphadenopathy and acquired immune deficiency syndromes. In Human T-Cell Leukemia/Lymphoma Virus, R. C. Gallo, M. Essex and L. Gross, eds. (Cold spring Harbor, N.Y.: Cold Spring Harbor Laboratory) pp. 363-370. Other human retroviruses, specifically two subgroups of the human t-cell leukemia/lymphoma/lymphotropic virus, types I and III have been isloated. (HTLV-I: Poicsz, B. J. et al. PNAS (USA) 77, 7415 (1980)); (HTLV-III: Popovic, M. et al. Detection, isolation and continuous production of cytopathic retroviruses (HTLV-III) from patients with AIDS and pre-AIDS. Science 224, 797-500 (1984)). Still another virus, the AIDS associated retrovirus (ARV), was proposed as the causative agent. Levy, J. A. et al. Isolation of lymphocytopathic retroviruses from San Francisco patients with AIDS. Science 225, 840-842 (1984)). Both the HTLV-III and ARV retroviruses display biological and sero-eidemiological properties similar to LAV. Levy et al., supra, Popovic et al. supra. As seen from the above, at least three (3) retroviruses have been postulated as the etiologic agent or AIDS: LAV; ARV; and, HTLV subtypes I and III.
LAV, HTLV-III and ARV-II genomes have been molecularly cloned. Shaw, G. M. et al., Serological analysis of a subgroup of human T-lymphotropic retroviruses (HTLV-III) associated with AIDS. Science 224, 503-505 (1984). Alizon, M. et al. Molecular Cloning of lymphadenopathy - associated virus. Nature, in press. The complete nucleotide sequence of the proviral genome of LAV, ARV and HTLV-III has been determined. Ratner, L. et al. Complete nucleotide sequence of the AIDS virus, HTLV-III. Nature 313, 277-284 (1985); Sanchez-Pescadov, R. et al. Nucleotide sequence and expression of an AIDS-associated retrovirus (ARV-2). Science 227, 484-492 (1985); and, Wain-Hobson, S. et al. Nucleotide sequence of the AIDS virus, LAV. Cell 40, 9-17 (1985).
One reason for the difficulty in determining the etiologic agent of AIDS was due to the reactivity of various retroviral antigens with serum samples from AIDS patients. For example, serum samples from AIDS patients have been shown to react with antigens of HTLV-I and HTLV-III. (HTLV-I: Essex, M., et al., "Antibodies to Cell Membrane Antigens Associated with Human T-Cell Leukemia Virus in Patients with AIDS", Science 220, 859(1983)); (HTLV-III: Sarngadharan, M. G. et al., "Antibodies Reactive With Human T-Lymphotropic Retroviruses (HTLV-III) in the Serum of Patients With AIDS", Science 224, 506-508 (1984)). Envelope gene products of HTLV demonstrated antigenicities cross-reactive with antibodies in sera from adult T-cell leukemia patients. Kiyokana, T. et al. Envelope proteins of human T-cell leukemia virus: Expression in Escherichia coli and its application to studies of env gene "functions" PNAS (USA) 81, 6202-6206 (1984). Adult T-cell leukemias (ATL) differ from acquired immune deficiency syndrome (AIDS) in that HTLV-I causes T-cell malignancies, that is uncontrolled growth of T-cell. In AIDS rather than cell growth there is cell death. In fact this cytopathic characteristic of HTLV-III was critical to determining ultimately the specific retroviral origin of the disease. Thus the etiologic agent of AIDS was isolated by use of immortalized human neoplastic T cell lines (HT) infected with the cytopathic retrovirus characteristic of AIDS, isolated from AIDS afflicted patients. Seroepidemiological assays using this virus showed a complete correlation between AIDS and the presence of antibodies to HTLV-III antigens. Gallo et al. supra 1984; Sarngadharan et al. supra 1984; Schupbach et al. Serological Analysis of a subgroup of human T-lymphotropic retroviruses (HTLV-III) associated with AIDS, Science 224, 503-505 (1984). In addition, nearly 85% of patients with lymphadenopathy syndrome and a significant proportion of asymptomatic homosexual men in AIDS endemic areas were also found to carry circulating antibodies to HTLV-III. Taken together, all these data indicate HTLV-III to be the etiologic agent for AIDS.
Until the successful culturing of AIDS virus using H-9 cell line the env AIDS protein of the AIDS virus had not been isolated, characterized or synthesized. This, in a major part, is due to the fact that the virus is cytopathic and thus isolation of the virus was not possible. Popovic M. et al., Detection, Isolation, and Continuous Production of Cytopathic Retroviruses (HTLV-III) From Patients With AIDS and Pre AIDs, Science 224, 497-500 (1984). Once the human T-cell line resistant to the cytopathic effects of the virus was discovered, a molecular clone of proviral DNA could be achieved.
The need for a sensitive and rapid method for the diagnosis of AIDS in human blood and its prevention by vaccination is very great. Virtually all the assays/tests presently available are fraught with errors. In fact the Center for Disease Control (CDC) has indicated that presently available tests be used solely for screening units of blood for antibody to HTLV-III. The CDC went further by stating that the presently available ELISA tests not be used for general screening of high risk populations or as a diagnostic test for AIDS. Federal Register 50(48), 9909, March 12, 1985. The errors have been traced to the failure to use a specific antigenic protein of the etiologic agent for AIDS. The previously used proteins were derived from a viral lysate. Since the lysate is made from human cells infected with the virus, i.e. the cells used to grow the virus, the lysate will contain human proteins as well as viral proteins. Thus preparation of a pure antigen of viral protein is very difficult. The antigen used produced both false positive and false negative results. Budiansky, S., AIDS Screening, False Test Results Raise Doubts, Nature 313, 583(1984).
The errors caused by the use of such lysate proteins/peptides can be avoided by using a composition for binding AIDS antibodies which is substantially free of the non-AIDS specific proteins. Compositions that are substantially pure HTLV-III env peptide fragments of the present invention can be used as antigens. The HTLV-III env peptides of the instant invention encompass a highly conserved epitope sequence designated HTLV-III env (500-511) which permit their use to screen for, diagnose and/or prevent by vaccination of the AIDS virus.
A synthetic peptide representing HTLV-III env (477-491) and its use as an immunogen for production of monoclonal antibodies which specifically bind to the envelope region of HTLV-III and which are used in a diagnostic reagent in an enzyme linked immunosorbent assay are described in U.S. Patent Application Ser. No. 779,431, filed Sept. 24, 1985, title HTLV-III/LAV Synthetic Peptide, Inventors F. Wong-Staal et al.